Deficiency in hepatic ATP-citrate lyase affects VLDL-triglyceride mobilization and liver fatty acid composition in mice
2010; Elsevier BV; Volume: 51; Issue: 9 Linguagem: Inglês
10.1194/jlr.m003335
ISSN1539-7262
AutoresQiong Wang, Shoufeng Li, Lei Jiang, Yunhua Zhou, Zi Li, Mengle Shao, Wenjun Li, Yong Liu,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoATP-citrate lyase (ACL) is a key lipogenic enzyme that converts citrate in the cytoplasm to acetyl-CoA, the initial precursor that yields malonyl-CoA for fatty acid biosynthesis. As cytosolic citrate is derived from the tricarboxylic acid cycle in the mitochondrion, ACL catalyzes a critical reaction linking cellular glucose catabolism and lipid synthesis. To investigate the metabolic action of ACL in lipid homeostasis, we specifically knocked down hepatic ACL expression by adenovirus-mediated RNA interference in mice maintained on a low-fat or high-fat diet. Hepatic ACL abrogation markedly reduced the liver abundance of both acetyl-CoA and malonyl-CoA regardless of dietary fat intake, which was paralleled with decreases in circulating levels of triglycerides and free fatty acids. Moreover, hepatic ACL knockdown resulted in diet-dependent changes in the expression of other lipogenic enzymes, accompanied by altered fatty acid compositions in the liver. Interestingly, ACL deficiency led to reduced serum VLDL-triglyceride levels but increased hepatic triglyceride content, resulting at least partially from decreased hepatic secretion of VLDL-containing apolipoprotein B-48. Together, these results demonstrate that hepatic ACL suppression exerts profound effects on triglyceride mobilization as well as fatty acid compositions in the liver, suggesting an important role for ACL in lipid metabolism. ATP-citrate lyase (ACL) is a key lipogenic enzyme that converts citrate in the cytoplasm to acetyl-CoA, the initial precursor that yields malonyl-CoA for fatty acid biosynthesis. As cytosolic citrate is derived from the tricarboxylic acid cycle in the mitochondrion, ACL catalyzes a critical reaction linking cellular glucose catabolism and lipid synthesis. To investigate the metabolic action of ACL in lipid homeostasis, we specifically knocked down hepatic ACL expression by adenovirus-mediated RNA interference in mice maintained on a low-fat or high-fat diet. Hepatic ACL abrogation markedly reduced the liver abundance of both acetyl-CoA and malonyl-CoA regardless of dietary fat intake, which was paralleled with decreases in circulating levels of triglycerides and free fatty acids. Moreover, hepatic ACL knockdown resulted in diet-dependent changes in the expression of other lipogenic enzymes, accompanied by altered fatty acid compositions in the liver. Interestingly, ACL deficiency led to reduced serum VLDL-triglyceride levels but increased hepatic triglyceride content, resulting at least partially from decreased hepatic secretion of VLDL-containing apolipoprotein B-48. Together, these results demonstrate that hepatic ACL suppression exerts profound effects on triglyceride mobilization as well as fatty acid compositions in the liver, suggesting an important role for ACL in lipid metabolism. As a major site for energy storage, processing, and conversion, the liver is a critical organ in the homeostatic control of both carbohydrate and lipid metabolism in mammals. De novo lipogenesis, which consists of a series of sequential reactions catalyzed by lipogenic enzymes (1Wakil S.J. Stoops J.K. Joshi V.C. Fatty acid synthesis and its regulation.Annu. Rev. Biochem. 1983; 52: 537-579Crossref PubMed Google Scholar), is a key metabolic process in the liver that contributes to the coordinate partitioning of energy fuels in response to changes in body's nutritional cues. Fatty acid biosynthesis is initiated in the cytosol with the generation of acetyl-CoA (CoA) from citrate that is transported out of the mitochondrion via the tricarboxylate transport system (2Kaplan R.S. Mayor J.A. Johnston N. Oliveira D.L. Purification and characterization of the reconstitutively active tricarboxylate transporter from rat liver mitochondria.J. Biol. Chem. 1990; 265: 13379-13385Abstract Full Text PDF PubMed Google Scholar). ATP-citrate lyase (ACL) (3Srere P.A. The citrate cleavage enzyme. I. Distribution and purification.J. Biol. Chem. 1959; 234: 2544-2547Abstract Full Text PDF PubMed Google Scholar) catalyzes this crucial step that links cellular glucose catabolism and fatty acid synthesis. Further conversion of acetyl-CoA to malonyl-CoA is catalyzed by acetyl-CoA carboxylase (ACC), which is the rate-limiting reaction in de novo fatty acid synthesis (4Thampy K.G. Koshy A.G. Purification, characterization, and ontogeny of acetyl-CoA carboxylase isozyme of chick embryo brain.J. Lipid Res. 1991; 32: 1667-1673Abstract Full Text PDF PubMed Google Scholar). Fatty acids are synthesized from malonyl-CoA through processes catalyzed by fatty acid synthase (FAS), long-chain elongase (ELOVL-6), and stearoyl-CoA desaturase-1 (SCD1) (5Ntambi J.M. Miyazaki M. Recent insights into stearoyl-CoA desaturase-1.Curr. Opin. Lipidol. 2003; 14: 255-261Crossref PubMed Scopus (207) Google Scholar). Saturated or unsaturated fatty acids are utilized to produce triglycerides (TG) by glycerol-3-phosphate acyltransferase (GPAT) and diacylglycerol acyltransferase (DGAT), which can be mobilized and transported from the liver primarily in the form of VLDL into circulation (6Havel R.J. Kane J.P. Quantification of triglyceride transport in blood plasma: a critical analysis.Fed. 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For instance, it has been reported that liver-specific ACC1 knockout mice (LACC1KO), while showing decreased liver TG contents with unchanged serum TG levels under fed states, exhibit no alterations in glucose homeostasis but much lower accumulation of lipids in the liver when fed a fat-free diet for 28 days (21Mao J. DeMayo F.J. Li H. Abu-Elheiga L. Gu Z. Shaikenov T.E. Kordari P. Chirala S.S. Heird W.C. Wakil S.J. Liver-specific deletion of acetyl-CoA carboxylase 1 reduces hepatic triglyceride accumulation without affecting glucose homeostasis.Proc. Natl. Acad. Sci. USA. 2006; 103: 8552-8557Crossref PubMed Scopus (219) Google Scholar). Unlike LACC1KO mice, however, mice with ablation of FAS in the liver (FASKOL mice) fed a zero-fat diet for 28 days develop fatty liver and hypoglycemia with decreased ketone bodies (22Chakravarthy M.V. Pan Z. Zhu Y. Tordjman K. Schneider J.G. Coleman T. Turk J. Semenkovich C.F. "New" hepatic fat activates PPARalpha to maintain glucose, lipid, and cholesterol homeostasis.Cell Metab. 2005; 1: 309-322Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). Whereas hepatic FAS ablation dramatically increases malonyl-CoA accumulation, ACC1 deficiency in the liver results in decreased production of malonyl-CoA, which is known as a crucial physiological inhibitor of lipid β-oxidation (21Mao J. DeMayo F.J. Li H. Abu-Elheiga L. Gu Z. Shaikenov T.E. Kordari P. Chirala S.S. Heird W.C. Wakil S.J. Liver-specific deletion of acetyl-CoA carboxylase 1 reduces hepatic triglyceride accumulation without affecting glucose homeostasis.Proc. Natl. Acad. Sci. USA. 2006; 103: 8552-8557Crossref PubMed Scopus (219) Google Scholar). In addition, liver-specific knockout of SCD-1 (LSCD1KO mice), which catalyzes the production of various unsaturated fatty acids, leads to protection of mice from adiposity and hepatic steatosis induced by a high-sucrose, very low-fat diet, but not by a high-fat diet (19Miyazaki M. Flowers M.T. Sampath H. Chu K. Otzelberger C. Liu X. Ntambi J.M. Hepatic stearoyl-CoA desaturase-1 deficiency protects mice from carbohydrate-induced adiposity and hepatic steatosis.Cell Metab. 2007; 6: 484-496Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). These studies all point to distinct metabolic consequences resulting from deficiency in each of the lipogenic enzymes, especially under different nutritional conditions.As a key lipogenic enzyme, ACL is most abundantly expressed in the liver and white adipose tissues, while exhibiting low expression levels in the brain, heart, small intestine, and muscles (23Fukuda H. Katsurada A. Iritani N. Effects of nutrients and hormones on gene expression of ATP citrate-lyase in rat liver.Eur. J. Biochem. 1992; 209: 217-222Crossref PubMed Scopus (44) Google Scholar). Global ACL ablation in mice has been shown to result in embryonic lethality, indicative of its essential role in embryonic development (24Beigneux A.P. Kosinski C. Gavino B. Horton J.D. Skarnes W.C. Young S.G. ATP-citrate lyase deficiency in the mouse.J. Biol. Chem. 2004; 279: 9557-9564Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). With respect to ACL's cellular functions, several groups have recently demonstrated that knockdown of ACL expression can suppress the proliferation and survival of several tumor cell lines with high glycolytic activities, suggesting that ACL, as an essential molecule in utilizing carbohydrates to provide carbon sources for cellular lipid synthesis required for tumor cell growth, represents a promising target for developing antitumor therapeutics (25Bauer D.E. Hatzivassiliou G. Zhao F. Andreadis C. Thompson C.B. ATP citrate lyase is an important component of cell growth and transformation.Oncogene. 2005; 24: 6314-6322Crossref PubMed Scopus (394) Google Scholar, 26Hatzivassiliou G. Zhao F. Bauer D.E. Andreadis C. Shaw A.N. Dhanak D. Hingorani S.R. Tuveson D.A. Thompson C.B. ATP citrate lyase inhibition can suppress tumor cell growth.Cancer Cell. 2005; 8: 311-321Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar, 27Migita T. Narita T. Nomura K. Miyagi E. Inazuka F. Matsuura M. Ushijima M. Mashima T. Seimiya H. Satoh Y. et al.ATP citrate lyase: activation and therapeutic implications in non-small cell lung cancer.Cancer Res. 2008; 68: 8547-8554Crossref PubMed Scopus (282) Google Scholar). Animal studies have also shown that pharmacological inhibition of ACL at the whole-body level results in hypolipidemia and leads to prevention of high-fat diet-induced weight gain and hyperlipidemia (28Pearce N.J. Yates J.W. Berkhout T.A. Jackson B. Tew D. Boyd H. Camilleri P. Sweeney P. Gribble A.D. Shaw A. et al.The role of ATP citrate-lyase in the metabolic regulation of plasma lipids. Hypolipidaemic effects of SB-204990, a lactone prodrug of the potent ATP citrate-lyase inhibitor SB-201076.Biochem. J. 1998; 334: 113-119Crossref PubMed Scopus (101) Google Scholar, 29Preuss H.G. Rao C.V. Garis R. Bramble J.D. Ohia S.E. Bagchi M. Bagchi D. An overview of the safety and efficacy of a novel, natural(-)-hydroxycitric acid extract (HCA-SX) for weight management.J. Med. 2004; 35: 33-48PubMed Google Scholar, 30Li J.J. Wang H. Tino J.A. Robl J.A. Herpin T.F. Lawrence R.M. Biller S. Jamil H. Ponticiello R. Chen L. et al.2-Hydroxy-N-arylbenzenesulfonamides as ATP-citrate lyase inhibitors.Bioorg. Med. Chem. Lett. 2007; 17: 3208-3211Crossref PubMed Scopus (53) Google Scholar). Similar to other lipogenic enzymes, ACL is also nutritionally regulated in response to changes in body's energy status. It has been reported that hepatic ACL in rodents displays relatively low expression levels in the state of starvation, whereas it is dramatically upregulated upon refeeding, with most prominent induction seen when fed a high-carbohydrate, low-fat diet (31Kornacker M.S. Lowenstein J.M. Citrate and the conversion of carbohydrate into fat. The activities of citrate-cleavage enzyme and acetate thiokinase in livers of starved and re-fed rats.Biochem. J. 1965; 94: 209-215Crossref PubMed Scopus (75) Google Scholar). Currently, it remains largely unclear to what extent hepatic ACL affects lipid metabolism in response to changes in body's nutritional states.Previously we have demonstrated that ACL in the liver, which is found to be markedly suppressed in mice fed a high-fat diet (13Jiang L. Wang Q. Yu Y. Zhao F. Huang P. Zeng R. Qi R.Z. Li W. Liu Y. Leptin contributes to the adaptive responses of mice to high-fat diet intake through suppressing the lipogenic pathway.PLoS ONE. 2009; 4: e6884Crossref PubMed Scopus (66) Google Scholar), acts as a critical component in mediating leptin's metabolic functions in regulating lipid and glucose metabolism (32Wang Q. Jiang L. Wang J. Li S. Yu Y. You J. Zeng R. Gao X. Rui L. Li W. et al.Abrogation of hepatic ATP-citrate lyase protects against fatty liver and ameliorates hyperglycemia in leptin receptor-deficient mice.Hepatology. 2009; 49: 1166-1175Crossref PubMed Scopus (153) Google Scholar). In leptin receptor-deficient db/db mice where hepatic ACL is abnormally elevated, targeted suppression of ACL in the liver can prominently correct obesity-associated fatty liver and hyperglycemia (32Wang Q. Jiang L. Wang J. Li S. Yu Y. You J. Zeng R. Gao X. Rui L. Li W. et al.Abrogation of hepatic ATP-citrate lyase protects against fatty liver and ameliorates hyperglycemia in leptin receptor-deficient mice.Hepatology. 2009; 49: 1166-1175Crossref PubMed Scopus (153) Google Scholar). To further understand the physiological contribution of hepatic ACL to lipid homeostasis, we investigated the metabolic consequences of hepatic ACL deficiency in mice under different nutritional conditions.EXPERIMENTAL PROCEDURESAnimal studiesC57BL/6 male mice (Shanghai Laboratory Animal Co. Ltd) were housed in laboratory cages at a temperature of 23 ± 3°C and a humidity of 35 ± 5% under a 12 h dark/light cycle (lights on at 6:30 a.m.) in accredited animal facilities at the Shanghai Institutes for Biological Sciences, CAS. Mice maintained on normal chow diet were infected with the adenoviruses and then fed ad libitum a low-fat diet (LFD, 10 kcal% fat) or high-fat diet (HFD, 60 kcal% fat) (Research Diets, Inc., New Brunswick, NJ). Total body fat content was measured by nuclear magnetic resonance (NMR) using the Minispec Mq7.5 (Bruker, Germany). Oxygen consumption and motility were determined for mice fed ad libitum in the comprehensive laboratory animal monitoring system (CLAMS, Columbus Instruments) according to the manufacturers' instructions. After acclimation to the system for 6 h, O2 and CO2 were measured for the following 24 h (through a 12 h light/dark cycle). Oxygen consumption was normalized to lean mass, and respiratory exchange ratio (RER) was calculated. Voluntary activity was determined by monitoring the X-axis beam breaks every 15 min. All animals were euthanized under anesthetic conditions. Livers were snap-frozen in liquid nitrogen immediately after resection and stored at −80°C. All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, CAS.Generation and administration of recombinant adenovirusesRecombinant adenoviruses Ad-shACL and Ad-shLacZ, which express shRNAs directed against ACL and LacZ genes, respectively, were generated using the BLOCK-iT™ Adenoviral RNAi Expression System (Invitrogen, Carlsbad, CA) as previously described (32Wang Q. Jiang L. Wang J. Li S. Yu Y. You J. Zeng R. Gao X. Rui L. Li W. et al.Abrogation of hepatic ATP-citrate lyase protects against fatty liver and ameliorates hyperglycemia in leptin receptor-deficient mice.Hepatology. 2009; 49: 1166-1175Crossref PubMed Scopus (153) Google Scholar). The two adenoviruses used for ACL knockdown contain the following target sequences: 5′-GCTGAATACCGAGGACATTAA-3′ for Ad-shACL1# and 5′-GCATTAAGCCTGGATGCTTTA-3′ for Ad-shACL4#, respectively. High-titer stocks of amplified recombinant adenoviruses were purified, and viral titers were determined by the tissue culture infectious dose 50 (TCID50) method. Viruses were diluted in PBS and administration was performed through tail-vein injection at approximately 5 × 108 pfu/mouse.Blood and liver measurementsHepatic triglyceride levels were measured by using 40–50 mg of liver tissue homogenized in 1.5 ml of a mixture of CHCl3-CH3OH (2:1, v/v), followed by shaking at room temperature for 2 h. After addition of 0.5 ml of 0.1 M NaCl, the suspension was centrifuged at 3,700 rpm for 10 min, and the lower organic phase was transferred and air-dried in a chemical hood overnight. The residual liquid was resuspended in 400 µl of 1% Triton X-100 in absolute ethanol, and the concentrations of triglyceride and cholesterol were analyzed using the commercial kits for blood measurements. Blood glucose was measured by a glucometer (FreeStyle, Alameda, CA). Serum and liver levels of triglycerides, FFA, β-hydroxybutyrate, and alanine transaminase (ALT) were determined using the Serum Triglyceride Determination Kit (Sigma, St. Louis, MO), FFA Half-micro Test (Roche Applied Science, Penzberg, Germany), β-Hydroxybutyrate LiquiColor (Stanbio, Boerne, TX) and Alanine Transaminase Determination Kit (ShenSuoYouFu, Shanghai, China), respectively.Determination of ACL activityACL enzyme activity was determined using the malate dehydrogenase (MDH)-coupled method as described (32Wang Q. Jiang L. Wang J. Li S. Yu Y. You J. Zeng R. Gao X. Rui L. Li W. et al.Abrogation of hepatic ATP-citrate lyase protects against fatty liver and ameliorates hyperglycemia in leptin receptor-deficient mice.Hepatology. 2009; 49: 1166-1175Crossref PubMed Scopus (153) Google Scholar). Briefly, liver extracts were incubated in the reaction buffer containing 20 mM citrate, 10 mM MgCl2, 10 mM DTT, 0.5 U/ml malic dehydrogenase, 0.33 mM CoASH, 0.14 mM NADH, and 100 mM Tris (pH 8.7), with or without 5 mM ATP. The yield of oxaloacetate generated by ACL catalysis was measured as the change in absorbance at 340 nm resulting from the consumption of NADH by the MDH-catalyzed reaction at 37°C. After subtracting the background changes, relative ACL activities were calculated by normalization to the total protein abundance of the extracts.Antibodies and Western immunoblot analysisACL and ACC antibodies were from Cell Signaling, Boston, MA; Monoclonal FAS antibody was purchased from BD Biosciences, San Jose, CA; GAPDH antibody was from KangChen, Shanghai, China; apoB antibody was from RayBiotech, Norcross, GA. For Western immunoblot analysis, tissue extracts were prepared by lysis with CelLytic™ MT (Sigma, St. Louis, MO) and centrifuged for 20 min at 20,000 g to remove the debris. Proteins (20–40 μg) from liver extracts or fast-protein liquid chromatography (FPLC) fractionation samples were separated by SDS-PAGE and transferred to PVDF filter membrane (Amersham Biosciences, Piscataway, NJ), which was subsequently subjected to immunoblotting with the desired antibodies.Real-time quantitative RT-PCRTotal liver RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA). After treatment with RNase-free DNase I (Roche Applied Science, Penzberg, Germany), first-strand cDNA was synthesized with M-MLV reverse transcriptase and random hexamer primers (Invitrogen). Real-time quantitative PCR was performed with the SYBR Green PCR system (Applied Biosystems, Foster City, CA), using GAPDH as an internal control for normalization. Primers used for each target gene were as follows: ACL, 5′-TGGATGCCACAGCTGACTAC-3′ and 5′-GGTTCAGCAAGGTCAGCTTC-3′; ACC1, 5′-TGAATCTCACGCGCCTACTATG-3′ and 5′-ATGACCCTGTTGCCTCCAAAC-3′; FAS, 5′AAGTTGCCCGAGTCAGAGAA-3′ and 5′-CGTCGAACTTGGAGAGATCC-3′; SCD1, 5′-GCGATACACTCTGGTGCTCA-3′ and 5′-CCCAGGGAAACCAGGATATT-3′; AceCS1, 5′-CGGACAGAGGGTGGCTATTA-3′ and 5′-AGGGTACCCAATGACAGCAG-3′; AceCS2, 5′-ATCCCCACATACCCAGATGA-3′ and 5′-GGTGCCGTGTAGAACTTGGT-3′; DGAT1, 5′-GTGTGTGGTGATGCTGATCC-3′ and 5′-GATGCAATAATCACGCATGG-3′; DGAT2, 5′-AGGCCCTATTTGGCTACGTT-3′ and 5′-GATGCCTCCAGACATCAGGT-3′; ELOVL6, 5′-TGCCATGTTCATCACCTTGT-3′ and 5′-TGCTGCATCCAGTTGAAGAC-3′; apoB, 5′-CAAGCACCTCCGAAAGTA-3′ and 5′-CACGGTATCCAGGAACAA-3′; apobec1, 5′-CGGGAGCTTCGGAAAGAGA-3′ and 5′-TCAACGTGGTTGCTGGTGTT-3.Quantification of acetyl-CoA and malonyl-CoAHomogenized liver tissues (20–25 mg) were added to the extraction solution (5% 5-sulfosalicylic acid containing 50 μM 1,4-dithioerythritol) at a ratio of 1:10 (w/v). After centrifugation at 14,000 g for 15 min at 4°C, the tissue homogenates were subjected to analysis by HPLC (Agilent 1200) and tandem mass spectrometry using negative-ion mode electrospray ionization with a 4000 Q-Trap system (Applied Biosystems, Foster City, CA) as previously described (32Wang Q. Jiang L. Wang J. Li S. Yu Y. You J. Zeng R. Gao X. Rui L. Li W. et al.Abrogation of hepatic ATP-citrate lyase protects against fatty liver and ameliorates hyperglycemia in leptin receptor-deficient mice.Hepatology. 2009; 49: 1166-1175Crossref PubMed Scopus (153) Google Scholar). The molecular ion peaks [M-H]− for acetyl-CoA, malonyl-CoA, and the internal standard n-propionyl-CoA were at m/z 808.3, 852.3 and 822.4, and their multiple reaction monitoring (MRM)-selected ions were 808.3/408.2, 852.3/ 408.2 and 822.4/408.3, respectively.Quantitative profiling of hepatic fatty acidsA modified Bligh and Dyer protocol was used to extract total lipids from the liver homogenates with a chloroform-methanol-water (2:1:0.8) mixture (33Sheaff R.C. Su H.M. Keswick L.A. Brenna J.T. Conversion of alpha-linolenate to docosahexaenoate is not depressed by high dietary levels of linoleate in young rats: tracer evidence using high precision mass spectrometry.J. Lipid Res. 1995; 36: 998-1008Abstract Full Text PDF PubMed Google Scholar). Freshly prepared 1,2-diheptadecanoyl-sn-glycero-phosphocholine (17:0) (Avanti Polar Lipids, Inc., AL) was added as an internal standard before homogenization. The extracts were saponificated using sodium hydroxide, followed by esterification with boron-trifluoride (BF3) (Sigma) in methanol to generate fatty acid methyl esters (FAME). Purified FAMEs were subsequently dissolved in heptane and stored in −20°C for further analysis.Total FAMEs were analyzed by gas chromatography-mass spectrometry (GC-MS) with a flame ionization detector (Agilent 5975B Inert XL MSD with 6890N GC; SP-2560 capillary column: 100 m × 0.25 mm I.D. × 0.20 μm film, Supelco, Inc., PA). Quantitative profiles were calculated using methyl-17:0 as the internal standard and an equal weight FAME mixture 68A (Nuchek Prep, Elysian, MN) as the response factor for each FAME. The amount of each fatty acid was calculated as relative to the wet weight of liver.Hepatic triglyceride secretion rate and lipoprotein fractionationHepatic triglyceride secretion rates were determined by measuring the increases in serum triglycerides after inhibition of lipoprotein lipase via tail-vein injection of tyloxapol (Sigma-Aldrich, St. Louis, MO) at 600 mg/kg, a dose that was supposed to completely inhibit triglyceride clearance during VLDL secretion experiments (34Lam T.K.T. Gutierrez-Juarez R. Pocai A. Bhanot S. Tso P. Schwartz G.J. Rossetti L. Brain glucose metabolism controls the hepatic secretion of triglyceride-rich lipoproteins.Nat. Med. 2007; 13: 171-180Crossref PubMed Scopus (120) Google Scholar). Serum samples were taken from the tail vein every h for triglyceride analysis.For lipoprotein fractionation analysis, equal volumes of serum samples were pooled from mice for each group in the fed states (a total volume of 400 μl). Lipoproteins were fractionated using a Superose 6 10/300 GL FPLC column (Amersham Biosciences, Piscataway, NJ). Fractions (500 μl) were collected and used for triglyceride and cholesterol analysis. For detection of apoB100 and apoB48, aliquots of the FPLC elution fractions were mixed with protein loading buffer and heated at 95°C for 6 min before Western immunoblot analysis.Analysis of apoB mRNA editingFor the measurement of C to U editing efficiency of apoB mRNA in the liver, a gel-purified 537-bp RT-PCR product harboring the editing site was subjected to direct sequencing. The primers used for PCR amplification of the editing region of apoB mRNA were: 5′-GCCCTGAGCAGACTTCCT-3′ and 5′-AATAGCGTCCATCTGTCG-3′. Editing efficiency was determined based on the T/C nucleotide signal ratio from the sequencing eletropherograms.HistologyLiver tissue specimens were fixed in 10% neutral buffered formalin, and then paraffin-embedded sections were subjected to standard hematoxylin-eosin (H and E) staining.Statistical analysisData are presented as means ± SEM. Differences were analyzed by unpaired two-tailed t-test between two groups or otherwise by one-way ANOVA.RESULTSHepatic ACL suppression results in hypotriglyceridemia with elevated liver triglyceride contentsPreviously we found that targeted suppression of dysregulated ACL in the liver of db/db mice dramatically reduced lipogenesis and led to markedly decreased levels of hepatic but not serum triglycerides (32Wang Q. Jiang L. Wang J. Li S. Yu Y. You J. Zeng R. Gao X. Rui L. Li W. et al.Abrogation of hepatic ATP-citrate lyase protects against fatty liver and ameliorates hyperglycemia in leptin receptor-deficient mice.Hepatology. 2009; 49: 1166-1175Crossref PubMed Scopus (153) Google Scholar). To further investigate the physiological importance of hepatic ACL in lipid homeostasis, we took the RNAi approach using two recombinant adenoviruses (Ad-shACL1# and 4#) that expressed shRNAs directed against two different coding regions of ACL. First we tested the effects of ACL knockdown in C57BL/6 mice maintained on a low-fat diet (10 kcal% fat). At 23 days post infection, both Ad-shACL1# and 4# efficiently knocked down the expression of ACL protein (supplementary ), resulting in a ∼77% reduction in the hepatic ACL enzyme activity compared with mice infected with the control virus Ad-shLacZ (supplementary ). In contrast to our observations in db/db mice (32Wang Q. Jiang L. Wang J. Li S. Yu Y. You J. Zeng R. Gao X. Rui L. Li W. et al.Abrogation of hepatic ATP-citrate lyase protects against fatty liver and ameliorates hyperglycemia in leptin receptor-deficient mice.Hepatology. 2009; 49: 1166-1175Crossref
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